Construction And Application Of Lysozyme/Lysozyme Fibrils Complex With Zein And Resveratrol

Proteins are the main undertakers and executors of life activities,and food proteins are important sources of essential nutrients for human life activities.Proteins can be divided into animal proteins and plant proteins depending on their source.Lysozyme is a common animal protein with advantages of good stability,small molecular weight and abundant sources.It is widely used as an antibacterial protein.Lysozyme fibrils（Ly-F）are the products of a large amount ofβ-sheet structure formed by lysozyme under extreme conditions.It has excellent mechanical properties,antibacterial activity,antioxidant activity,etc.Zein is a common plant protein containing a large number of hydrophobic amino acid residues and is often used as a carrier for delivery systems.Resveratrol（Res）is a natural hydrophobic polyphenol with antioxidant,anti-inflammatory,and other biological activities.But its usage in the food industry is greatly limited by its poor solubility,light and heat instability,and low bioavailability.The multifunctional and amphiphilic properties of proteins make them interact with each other or other components（polyphenols,polysaccharides,etc.）in food under certain conditions.These interactions can affect and determine food properties,such as texture characteristics and storage stability,etc.Therefore,revealing the interaction mechanism between animal protein and plant protein,or protein and polyphenol at the molecular level can provide an important theoretical basis for the development of innovative products and the design of bioactive substances carriers in food industry.In this thesis,the interaction mechanism between lysozyme and zein was studied by experimental method and molecular dynamics simulation.Lysozyme fibrils were prepared and the interaction mechanism between lysozyme fibrils and zein was explored.The lysozyme fibrils-zein complexes were prepared and used to encapsulate and protect resveratrol.The formation mechanism of resveratrol/lysozyme fibrils hydrogel was studied through experiments and molecular dynamics simulation.The main conclusions are as follows:（1）The interaction between Zein and Lysozyme was studied by experimental methods and molecular dynamics simulation.Zein-Lysozyme complex nanoparticles were prepared by anti-solvent precipitation method.The effects of the mass ratio of Zein and Lysozyme on the particle size,surface hydrophobicity,thermal stability and micro-morphology of the complex nanoparticles were investigated.The particle size experiment results showed that the particle size of complex nanoparticles was significantly lower（118.7～134.6 nm）than that of Lysozyme（360.7 nm）.The surface hydrophobicity and thermal stability experiments showed that Zein-Ly_2:1 hydrophobic region was the most exposed,and the thermal stability was the best.Transmission electron microscopy（TEM）images showed that the complex nanoparticles were aggregated,and field emission scanning electron microscopy（FE-SEM）images showed that the surface morphology of the complex nanoparticles was relatively rough.The results of dynamic light scattering（DLS）,Fourier transform infrared spectroscopy（FTIR）and differential scanning calorimetry（DSC）showed that there was hydrogen bonding,electrostatic and hydrophobic interactions between Zein and Lysozyme.The dissociation experiment results showed that hydrophobic interaction was the main driving force in Zein-Lysozyme complex nanoparticles,and hydrogen bonding also existed.The results of molecular dynamics simulation showed that Lysozyme was more inclined to combine with the convex region of Zein.Residues P192,A193,A194,Y195,L196,Q198,L199,N203,Q245,L253 of Zein and Residues D49,Y63,W64,A108,W109,V110 of Lysozyme were the key residues for the combination of Zein and Lysozyme.（2）Lysozyme fibrils（Ly-F）were prepared from lysozyme at p H 2.0 and 90℃ for 24 h.The UV-absorption spectrum showed that the maximum absorption peak of Ly-F combined with congo red（CR）shifted to 500 nm and a shoulder peak appeared at 540 nm.The fluorescence spectrum showed that the fluorescence intensity of Ly-F combined with thioflavin T（Th T）and 8-aniline-1-naphthalenesulfonic acid（ANS）increased significantly at 486 nm and480 nm,respectively.Atomic force microscopy（AFM）and TEM images showed that Ly-F had typical fibrils morphology.Ly-F/Zein complex nanoparticles were prepared by anti-solvent precipitation method,and the influence of mass ratio of Ly-F to Zein on the turbidity andζ-potential values of complex nanoparticles was investigated.The stability of Ly-F/Zein_1:2 was the best.The interaction mechanism of Ly-F and Zein was revealed by DLS,FTIR and dissociation experiments.The results showed that hydrogen bonding,electrostatic and hydrophobic interactions were involved in the formation of Ly-F/Zein complex nanoparticles.The complex nanoparticles were used to encapsulate resveratrol and the conditions of process were optimized.The results showed that the encapsulation efficiency of complex nanoparticles for resveratrol reached to the highest（82.58%）when the resveratrol concentration was 30μg/m L.X-ray diffraction（XRD）results showed that resveratrol was successfully encapsulated by Ly-F/Zein complex nanoparticles and had amorphous properties.The micromorphology,Uv and storage stabilities,solubility,antioxidation and antibacterial properties of Ly-F/Zein-Res complex nanoparticles were studied.The results showed that Ly-F/Zein-Res presented as a shape of"fruit tree"with dense fruit with good stability and biological activity.（3）Resveratrol/lysozyme fibril（Res/Ly-F）hydrogels were prepared by self-assembly method.The rheological properties,microscopic morphology,antibacterial and antioxidant activities of the hydrogels were measured.The interaction mechanism between resveratrol and lysozyme fibrils was explored by experiments and molecular dynamics simulation.The rheological results showed that the hydrogel with the molar ratio of resveratrol to Ly-F of 2:1had better gel properties.TEM showed that Res/Ly-F hydrogels had dense network.The results of UV-vis,FTIR,surface hydrophobicity（H₀）and dissociation experiments showed that hydrophobic,hydrogen bonding and electrostatic interactions were involved in the formation of hydrogels,and hydrophobic interaction was the main driving force.Molecular dynamics simulation results showed that the residues of N59,N46,D52 and W62 in Ly-F played a key role in the binding with resveratrol,and N59 was the key residue.The antioxidant activity of Res/Ly-F hydrogel was significantly higher than that of Ly-F and resveratrol,and the antioxidant activity of the hydrogel was concentration dependent.Res/Ly-F hydrogel also had significant antibacterial,and has potential application value in the field of biology and medicine.